Sensor chip for detecting binding kinetics between biomolecules as well as preparation method and application of sensor chip

Abstract

The invention provides a sensor chip for biomolecular binding kinetics detection and a preparation method and application thereof, and relates to the technical field of sensor chips, the sensor chip for biomolecular binding kinetics detection comprises a control substrate and a sensing substrate, the top end of the sensing substrate is bonded with the bottom end of the control substrate, and the top end of the sensing substrate is bonded with the bottom end of the control substrate. A first liquid accumulation tank covered by the reaction tank is dug in the top end of the sensing substrate, a structural film or a sensitive film is erected above the first liquid accumulation tank, a local modification area is arranged at the top end of the sensitive film, and four cantilever beams are uniformly distributed on the side walls of the structural film and the sensitive film in the circumferential direction and are fixedly connected with the inner wall of the first liquid accumulation tank; a piezoresistor is arranged at the top end of each cantilever beam, every two adjacent piezoresistors located above the same first liquid accumulation groove are connected through a lead to form a Wheatstone bridge, each lead is connected with a bonding pad, and the sensor chip manufactured through the method is high in sensitivity, high in resolution, small in temperature drift, low in cost and capable of achieving miniaturized detection.

Description

Technical Field

[0001] The present invention relates to the technical field of sensor chips, and in particular to a sensor chip for detecting binding kinetics between biological molecules, and a preparation method and application thereof. Background Art

[0002] The interaction between biomolecules such as proteins, DNA and small molecules is the basis for maintaining life activities. By studying the binding characteristics of these molecules, we can reveal the mechanisms of important biological processes such as cell signal transduction, enzyme catalysis, transcriptional regulation, etc. The detection of binding dynamics between biomolecules has important scientific and practical significance in the fields of biology, drug development, disease treatment, etc.

[0003] The kinetic measurement methods for biomolecular interactions mainly include fluorescence, weight and optical methods. Among them, the fluorescence method detects by directly binding to the analyte, which is particularly suitable for binding assessment of unconventional materials such as cell surfaces. However, this method requires the use of labeled probes to modify the ligand, which is not only time-consuming and labor-intensive, but may also interfere with the natural binding behavior of the target biomolecule, thereby affecting the accuracy of the measurement. In contrast, the weight and optical methods use the principle of non-label detection to avoid potential interference caused by probe modification. However, these two methods have high requirements for the size of the target molecule and lack the detection sensitivity for low molecular weight substances. It is worth noting that such technologies generally have equipment dependence problems at the application level-large and precise instruments are required, resulting in limited detection throughput and high R&D costs. These technical bottlenecks jointly restrict their application and promotion in conventional laboratories and high-throughput screening scenarios.

[0004] In recent years, more and more sensors based on micro-nano processing technology have been applied to the study of biomolecular interactions. Among them, biosensors based on surface stress are a common label-free biosensing technology. This surface stress-based biosensor uses the binding energy of chemical bonds between biomolecules to test the analyte. When biomolecules interact with recognition elements (such as antibodies, enzymes, DNA, etc.) on the sensor surface, stress changes on the sensor surface will be caused. This stress change can be detected and quantified by small deformations of the sensor structure or changes in physical properties such as capacitance and resistance. By monitoring the changes in these physical parameters, qualitative and quantitative analysis of biomolecules can be achieved. Related research mainly focuses on two structures, micro-cantilever beam type or micro-film type, which can be made into parallel array structures to achieve high-sensitivity simultaneous detection of multiple independent sensing units with different surface functionalizations. The patent with application number: 202010327183.3 discloses a design method for a hollow micro-nano composite beam for biochemical molecule detection. The design structure proposed by the method includes a beam body and a hollow microfluidic channel for biochemical reactions. The beam is composed of two structural materials: a silicon or silicon compound substrate and a flexible film. Between the flexible film layer and the silicon or silicon compound substrate layer, there are a liquid inlet, a liquid outlet, a liquid inlet microchannel, a liquid outlet microchannel and a reaction pool for the sample to be tested, and the reaction pool is located in the mass sensitive area of ​​the beam. This method proposes a "liquid reaction-vacuum detection" method, which adsorbs the object to be tested inside the microchannel and places the entire structure in a vacuum environment. This detection method can effectively isolate the reaction environment from the detection environment. By detecting the frequency changes caused by the reaction of the object to be tested, high-sensitivity, in-situ and real-time rapid detection of biochemical molecules is achieved, which can be widely used in engineering fields such as medicine and chemical industry. However, this method requires the use of a vacuum pump to evacuate it to obtain a vacuum environment, so its detection cost rises sharply.

[0005] Therefore, there is a need to provide a sensitive and efficient method for detecting biomolecule binding directly with electrical signals without the use of expensive additional equipment. Summary of the invention

[0006] The problem solved by the present invention is to provide a sensor chip for detecting binding kinetics between biological molecules and a preparation method and application thereof. The sensor chip has high sensitivity, high resolution, small temperature drift, low cost and can realize miniaturized detection.

[0007] The first aspect of the present invention provides a sensor chip for detecting binding kinetics between biomolecules, comprising: A control substrate, wherein at least one sample inlet and at least one sample outlet are provided on the control substrate, a reaction groove and two microchannels connected to the reaction groove are dug at the bottom end of the control substrate, and the reaction groove is connected to the sample inlet and the sample outlet respectively through the two microchannels; A sensing substrate, the top of the sensing substrate is bonded to the bottom of the control substrate, at least two first liquid accumulation grooves covered by the reaction groove are dug at the top of the sensing substrate, a structural film and a sensitive film are respectively mounted above the two first liquid accumulation grooves, a local modification area is provided at the top of the sensitive film, four cantilever beams are evenly distributed circumferentially on the side walls of the structural film and the sensitive film and are fixedly connected to the inner wall of the first liquid accumulation groove through the four cantilever beams, a piezoresistor is provided at the top of each cantilever beam, and each adjacent two piezoresistors above the same first liquid accumulation groove are connected through leads to form a Wheatstone bridge, and each lead is connected to a pad.

[0008] Compared with the prior art, the sensor chip for detecting the binding kinetics between biomolecules of the present invention has the following advantages: In the present invention, an independent piezoresistance is arranged on each cantilever beam, and adjacent piezoresistances are electrically connected through leads to form a Wheatstone bridge with self-compensation effect, a local modification area is arranged on the top of the sensitive film to form a stress concentration area, and an independent reaction tank and a microfluidic channel are integrated at the bottom of the control substrate. By covering each first liquid accumulation tank with the reaction tank to form a closed reaction chamber, the directional capture and localized control of the target molecule binding model when the sensor chip is used are ensured; and the fluid loop formed by the sample inlet, microfluidic channel, reaction tank, and sample outlet supports dynamic sample circulation and real-time flushing, which can not only maintain the stability of the reaction environment and avoid cross contamination, but also realize the automatic switching of multi-step detection processes, which significantly improves the detection throughput while taking into account the efficient utilization of trace samples and the consistent regulation of detection conditions.

[0009] Furthermore, the present invention uses a thin film cantilever beam array, and adopts a differential processing method to measure the results of the sensitive film with a local specific reaction biosensitive layer and the structural film without a specific reaction biosensitive layer, which effectively reduces the measurement error caused by nonspecific adsorption problems.

[0010] In a possible embodiment, each of the leads includes two sub-leads connected to each other at one end, each of the sub-leads includes a heavily doped lead and a metal lead connected to the heavily doped lead, and each two adjacent piezoresistors above the same first liquid accumulation groove are connected in sequence through the heavily doped lead and the metal lead.

[0011] Compared with the prior art, after adopting the above technical solution, the heavily doped lead is used as the first connecting segment and the metal lead is used as the second connecting segment. Due to the characteristics of the heavily doped lead doped with higher concentrations of impurity elements, the number of carriers that can be transmitted is significantly increased, thereby improving the conductive performance.

[0012] In a possible implementation manner, a second liquid accumulation groove is dug at the top of each of the structural film and the sensitive film, a through hole is provided in the middle of the local modification area, and the through hole is communicated with the second liquid accumulation groove on the sensitive film.

[0013] In a possible implementation, four first liquid collection grooves covered by the reaction groove are dug at the top of the sensing substrate, and a structural film is mounted above one of the first liquid collection grooves, and a sensitive film is mounted above the other three first liquid collection grooves.

[0014] Compared with the prior art, after adopting the above technical scheme, the baseline value test is carried out through the structural film of a single area, and the measured value test is carried out through the sensitive films of three areas. Compared with the setting of the sensitive film in a single area, the accuracy of the measured value can be further improved and the error influence caused by the change of a single parameter can be reduced.

[0015] Another object of the present invention is to provide a method for preparing a sensor chip, comprising the following steps: S1, selecting an SOI substrate including a silicon support layer, a buried oxide layer, and a silicon device layer from bottom to top; S2, sequentially performing photolithography, development and dry etching on the SOI substrate to obtain grooves on the surfaces of the silicon device layer and the buried oxide layer; S3, growing a first SiO on the surface of the groove and the silicon device layer 2 layer; S4, after sequentially performing photolithography and development, lightly doping with boron ions to obtain a piezoresistor, and then heavily doping with boron ions to obtain a heavily doped lead; S5, depositing a second SiO on the top and bottom surfaces respectively 2 layer; S6, after sequentially performing photolithography, development and dry etching, a lead hole is obtained at the end of the heavily doped lead; After sequentially performing metal sputtering, photolithography, development and etching, metal leads and pads are obtained, and ohmic contact is formed between the metal leads and the heavily doped leads; S7, selectively sputtering at least one metal layer on the top surface, and obtaining a sensitive film by a metal lift-off method; S8, deposit the third SiO 2 layer and a silicon nitride layer, and then photolithography and development to obtain a metal passivation layer; S9, after sequentially performing photolithography, development and dry etching on the top surface, the silicon support layer is exposed and a through hole is obtained in the local modified area to obtain a second liquid storage groove; S10, performing anisotropic wet etching to obtain a first liquid accumulation groove and a cantilever beam, thereby obtaining a sensing substrate; S11, using a flexible film to control the substrate; S12, bonding the control substrate and the sensing substrate to obtain a sensor chip.

[0016] Compared with the existing technology, this preparation method realizes high-precision manufacturing of sensor chips through the coordinated integration of SOI substrate and multiple processes: the etching and doping processes based on the SOI heterostructure accurately control the nanoscale morphology and piezoresistive properties of the cantilever beam to ensure the matching of mechanical and electrical properties; multi-step oxidation and passivation layer deposition enhance the interface stability and biocompatibility; the combination of wet etching and bonding processes ensures the integrity of the suspended structure while seamlessly integrating the microfluidic functional modules, taking into account the process repeatability and the reliable forming of complex three-dimensional structures, and providing an efficient and controllable technical path for the batch preparation of high-performance biosensor chips; not only that, the preparation processes provided by the present invention all adopt conventional and mature MEMS processing technologies, the method has low cost and high reliability, and can achieve large-scale batch manufacturing.

[0017] In a possible implementation, in the step S1, in the SOI substrate, the thickness of the silicon device layer is 2.9-3.1 μm, the thickness of the buried oxide layer is 0.9-1.1 μm, and the thickness of the silicon support layer is 690-710 μm.

[0018] Compared with the prior art, the present invention achieves the coordinated optimization of the device's mechanical properties and process feasibility by precisely matching the thickness ratio of each functional layer of the SOI substrate: the thickness of the silicon device layer ensures that the cantilever beam has both high-sensitivity deflection and fatigue resistance, the thickness of the buried oxide layer balances the etching accuracy and interface stress buffering effect, and the thickness of the silicon support layer provides sufficient mechanical stability for the subsequent release of the suspended structure. The three work together to ensure the structural integrity and process compatibility of the chip in nanoscale deformation sensing and microfluidic integration, laying the foundation for high-yield manufacturing.

[0019] In a possible implementation, in step S7, the material of the metal layer is a mixture of gold and chromium or a mixture of gold and titanium.

[0020] Compared with the prior art, the present invention takes into account both interface bonding strength and biofunctional adaptability through the design of a composite metal layer: the mixed use of gold and chromium or titanium not only utilizes the high adhesion of chromium / titanium to ensure the firm bonding of the metal layer to the silicon substrate, but also provides a stable, low-background interference active interface for subsequent biomolecule modification through the chemical inertness of gold, while optimizing the electrical signal transmission efficiency and environmental corrosion resistance, so that the sensing interface maintains long-term functional stability in a complex biochemical environment.

[0021] In a possible implementation manner, in step S8, the third SiO 2 The thickness of the layer is 195-205 nm, and the thickness of the silicon nitride layer is 95-105 nm.

[0022] Compared with the prior art, the present invention achieves dual optimization between mechanical protection and interface function by precisely controlling the thickness ratio of the passivation layer: 2 The silicon nitride layer provides flexible stress buffering and electrical insulation isolation, while the silicon nitride layer provides a highly chemically inert surface barrier. The two work together to form a gradient protection system, which can not only effectively resist the erosion and ion penetration of biological fluids, but also avoid the rigid constraints of the cantilever beam's micro-nano deformation caused by excessively thick coatings, thereby ensuring the stability of the sensing interface and the signal fidelity in long-term biochemical reactions.

[0023] In a possible implementation manner, the specific operations of step S11 are as follows: S11, taking a single-side polished single crystal silicon wafer, and subjecting it to photolithography and development treatments in sequence to obtain a silicon wafer mold; A mixed liquid containing a flexible membrane material and a curing agent is prepared, and a silicon wafer mold is placed in the mixed liquid until it solidifies. The mold is demolded, sliced ​​and punched in sequence to obtain a control substrate with an inlet, an outlet, a reaction groove and a microfluidic channel.

[0024] Compared with the prior art, the present invention achieves high-precision structural replication and biocompatibility assurance in the preparation of microfluidic control parts by combining single-crystal silicon molds with flexible material replica processes: the photolithography molding of the silicon mold ensures the strict controllability of the reaction chamber and flow channel dimensions, forming a spatial match with the cantilever beam unit of the sensor; the casting and curing process of the flexible material gives the control part excellent deformation adaptability, which not only simplifies the processing difficulty of complex three-dimensional flow channels, but also maintains the intrinsic activity of the biological reaction environment through chemically inert surface properties, providing an efficient solution for high-reliability chip packaging and fluid manipulation.

[0025] The third aspect of the present invention provides an application of a sensor chip in the detection of binding kinetics between biological molecules. The method has the characteristics of being simple, rapid, low-cost, and reusable, and can be widely used in the measurement of binding equilibrium constants and kinetic parameters of various types of molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of the explosion structure of the present invention; Figure 2 is a schematic structural diagram of the top of the sensing substrate of the present invention; Figure 3 It is a structural schematic diagram of the bottom end of the control substrate of the present invention; Figure 4 This is a schematic diagram of the structure of the central region of the top of the sensing substrate of the present invention; Figure 5 A schematic diagram of a partial structure of a first liquid collection tank area of ​​the present invention; Figure 6 A process flow chart for preparing a sensing substrate according to the present invention; Figure 7 is a cross-sectional view of the sensor chip of the present invention; Figure 8 This is one of the SEM scanning electron microscope images of the sensor chip of the present invention; Fig. 9 This is the second SEM scanning electron microscope image of the sensor chip of the present invention; Fig.10 This is the third SEM scanning electron microscope image of the sensor chip of the present invention; Fig.11 The SEM scanning electron microscope images of sensor chips of different geometric shapes prepared by the present invention; Description of reference numerals: 1. Control substrate; 11. Inlet; 12. Outlet; 13. Reaction tank; 14. Microfluidic channel; 2. Sensing substrate; 21. First liquid accumulation tank; 22. Structural film; 23. Sensitive film; 24. Cantilever beam; 25. Piezoresistance; 26. Lead; 261. Heavily doped lead; 262. Metal lead; 27. Pad; 28. Second liquid accumulation tank; 3. Local modification area. DETAILED DESCRIPTION

[0027] In order to make the above-mentioned purposes, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation method and typical parameters of the present invention, and are not used to limit the parameter range described in the present invention. Reasonable changes derived therefrom are still within the scope of protection of the claims of the present invention.

[0028] It should be noted that the endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

[0029] Unless otherwise defined, all terms, symbols and other scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms with conventionally understood meanings are defined herein for the purpose of clarification or ease of reference, and such definitions herein should not be construed as indicating significant differences from conventional understandings in the art. The technical methods described or cited herein are generally well understood by those skilled in the art and are adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents and instruments is carried out in accordance with the protocols and parameters given by the manufacturer.

[0030] See also Figure 1-Figure 5 The present application embodiment discloses a sensor chip for detecting binding kinetics between biomolecules, comprising a control substrate 1 and a sensing substrate 2 bonded to each other, wherein the control substrate 1 covers the top of the sensing substrate 2, wherein the control substrate 1 is provided with at least one injection port 11 and one outlet port 12, preferably two injection ports 11 in this embodiment, a microchannel 14 and a reaction tank 13 are provided at the bottom of the control substrate 1, the two injection ports 11 are connected to the reaction tank 13 through a microchannel 14 respectively, and the outlet port 12 is connected to the reaction tank 13 through a microchannel 14; at least two first liquid accumulation grooves 21 are provided in the middle of the top of the sensing substrate 2, and the two first liquid accumulation grooves 21 are provided in the middle of the top of the sensing substrate 2. The liquid tanks 21 are all covered by the reaction tank 13. A structural film 22 and a sensitive film 23 are respectively mounted above the two first liquid storage tanks 21, wherein a local modification area 3 is provided at the top of the sensitive film 23. Four cantilever beams 24 are evenly distributed circumferentially on the side walls of the structural film 22 and the sensitive film 23, and are fixedly connected to the inner wall of the first liquid storage tank 21 through the four cantilever beams 24. A piezoresistance 25 is provided at the top of each cantilever beam 24. Every two adjacent piezoresistances 25 above the same first liquid storage tank 21 are connected through a lead 26 to form a Wheatstone bridge. Each lead 26 is connected to a pad 27, and the input and output of electrical signals are realized through the pad 27.

[0031] In the embodiment of the present application, in order to further improve the detection effect, four first liquid accumulation grooves 21 are opened in the middle part of the top of the sensing substrate 2, and the four first liquid accumulation grooves 21 are arranged in a 2x2 array. A structural film 22 and a sensitive film 23 are mounted above the four first liquid accumulation grooves 21, wherein a sensitive film 23 is mounted above any three of the first liquid accumulation grooves 21, and a structural film 22 is mounted above the remaining first liquid accumulation groove 21, and a local modification area 3 is provided on the top of the sensitive film 23.

[0032] In the embodiment of the present application, the width of the microchannel 14 is 300 μm-500 μm, and the diameters of the sample inlet 11 and the sample outlet 12 are both 1 mm.

[0033] In the embodiment of the present application, the cross-sections of the structural film 22 and the sensitive film 23 are both annular octagons with a thickness of 2μm-4μm, and the width of the cantilever beam 24 is 80μm-140μm, wherein the bottom end of the cantilever beam 24 is at the same horizontal height as the top end of the structural film 22 or the sensitive film 23, that is, there is a height difference between the cantilever beam 24 and the local modification area 3, so that a stress concentration area is formed on the sensitive film 23, and each sensitive film 23 is provided with a local modification area 3, and the surface of the local modification area 3 is used to modify the biorecognition molecules.

[0034] In the embodiment of the present application, each lead 26 includes two sub-leads, each sub-lead includes a heavily doped lead 261 and a metal lead 262 connected to the heavily doped lead 261, and each adjacent two piezoresistors 25 above the same first liquid accumulation groove 21 are connected in sequence through the heavily doped lead 261 and the metal lead 262. A second liquid accumulation groove 28 is dug at the top of the structural film 22 and the sensitive film 23, and a through hole is provided in the middle of the local modification area 3, and the through hole is communicated with the second liquid accumulation groove 28 on the sensitive film 23.

[0035] like Figure 6 and Figure 7 As shown, a specific embodiment of the present invention further provides a method for preparing a sensor chip, and the preparation method specifically comprises the following steps: S1, using a (100) crystal plane SOI substrate, wherein the SOI substrate includes a silicon support layer, a buried oxide layer, and a silicon device layer from bottom to top; S2, sequentially subjecting the silicon device layer to photolithography, development and dry etching to obtain grooves on the surfaces of the silicon device layer and the buried oxide layer; S3, the groove obtained in step S2 and the surface of the silicon device layer are thermally oxidized to form a first SiO 2 layer; S4, the structure obtained in step S3 is sequentially subjected to photolithography and development, and then subjected to light boron ion doping to form a piezoresistor 25, and then subjected to heavy boron ion doping to form a heavily doped lead 261; S5, depositing a second SiO 2 layer; S6, based on the structure obtained in step S5, a lead hole is formed at the end of the heavily doped lead 261 by sequentially performing photolithography, development and dry etching processes; Then, metal lead 262 and pad 27 are obtained by metal sputtering, photolithography, development and etching in sequence, wherein metal lead 262 and heavily doped lead 261 are in ohmic contact by high temperature boron ion diffusion and annealing. S7, selectively sputtering at least one metal layer on the top surface of the structure obtained in step S6, and forming a sensitive film 23 by a metal lift-off method; S8, depositing a third SiO 2 layer and a silicon nitride layer, and then through photolithography and development processing to form a metal passivation layer; S9, the top surface of the structure obtained in step S8 is subjected to photolithography, development and dry etching in sequence to etch away the first SiO 2 Layer and second SiO 2 layer, exposing the silicon support layer of the SOI substrate and obtaining a through hole in the local modified area 3 to form a second liquid collection groove 28; S10, performing anisotropic wet etching on the structure obtained in step S9 to obtain a first liquid collection groove 21 and a cantilever beam 24, thereby completing the manufacturing of the sensor substrate 2; S11, manufacturing a sample inlet 11, a sample outlet 12, a reaction tank 13 and a microfluidic channel 14 on a flexible membrane having biocompatibility to obtain a control substrate 1; S12. Using a chemical bonding method, the control substrate 1 and the sensing substrate 2 are bonded to obtain a sensor chip.

[0036] In the embodiment of the present application, in step S1, in the SOI substrate, the thickness of the silicon device layer is 2.9-3.1 μm, the thickness of the buried oxide layer is 0.9-1.1 μm, and the thickness of the silicon support layer is 690-710 μm.

[0037] In the embodiment of the present application, in step S7, the material of the metal layer is a mixture of gold and chromium or a mixture of gold and titanium.

[0038] In the embodiment of the present application, in step S8, the third SiO 2 The thickness of the layer is 195-205nm, and the thickness of the silicon nitride layer is 95-105nm.

[0039] In the embodiment of the present application, the specific operation of step S11 is as follows: S11, taking a single-side polished single crystal silicon wafer, and sequentially performing photolithography and development processes to obtain a silicon wafer mold; A mixed liquid containing a flexible membrane material and a curing agent is prepared, and a silicon wafer mold is placed in the mixed liquid until it solidifies. The control substrate 1 having an inlet 11, an outlet 12, a reaction tank 13 and a microchannel 14 is obtained by sequentially performing demolding, slicing and punching processes.

[0040] Specifically, the volume lithography process, development process, dry etching process and thermal oxidation growth process in the preparation process of the present invention all adopt existing technologies. More specifically, the following parameters can be selected and adjusted according to the actual preparation process.

[0041] Photolithography: Spin coat the structure surface with photoresist (thickness 0.5-2μm) evenly, pre-bake (90-120℃, 1-5 minutes) and solidify, then use ultraviolet light source (such as 193nm DUV) to expose through the mask (dose 100-300 mJ / cm²), and then post-bake (110-130℃, 1-5 minutes) to enhance the pattern stability, and finally form a high-resolution (0.1-0.5 μm) photoresist pattern to provide a precise mask for subsequent etching.

[0042] Development: Use TMAH solution (positive photoresist) or special developer (negative photoresist) to remove the photoresist in the unexposed / exposed areas, control the development time (30-120 seconds) and temperature (20-25°C), use ultrasound to improve uniformity, accurately retain the pattern edge (roughness <1 nm), avoid undercut effect, and ensure high consistency with the mask pattern.

[0043] Dry etching: Reactive ion etching (RIE) or inductively coupled plasma (ICP-RIE) is used to etch the 6 / Cl 2 Mixed gas (ratio 50:10 sccm) is the main etchant, supplemented by O 2 Depolymerize and cool with He, set the RF power (50-300 W), bias voltage (-50 to -500 V) and low pressure (1-100 mTorr), transfer the photoresist pattern to the silicon and buried oxide layers through anisotropic etching (aspect ratio of 10:1-50:1), control the etching depth (e.g. 500 nm) by endpoint detection (OES monitors the SiF characteristic peak), and optimize the selectivity (Si / PR reaches 10:1-100:1) to avoid damage to the buried oxide layer.

[0044] Thermal Oxidation Growth: Thermal oxidation growth of the first SiO 2The steps of the layer include: firstly, removing the contaminants on the surface of the silicon wafer by RCA cleaning method and drying; then selecting dry oxygen (800-1000℃, O 2 )、Wet oxygen(700-900℃,H 2 O participation) or water vapor oxidation (direct steam), control temperature, time and gas flow (such as dry oxygen O 2 flow rate 1-10 slm) to adjust the thickness (10-500nm); after growth, the lattice defects are repaired by rapid thermal annealing (900-1100℃), and finally a low interface state density (<1×10 10 cm -2 eV -1 ), a dense oxide layer with high uniformity (±3%).

[0045] According to the above preparation method, the SEM scanning electron microscope image of the sensor chip prepared is as follows: Figure 8-Figure 10 As shown; Fig.11 The SEM images of sensor chips with different geometric shapes prepared by the inventors are shown.

[0046] The present invention also provides a method for detecting binding kinetics between biomolecules, comprising the following steps: Step A1, immobilizing the probe molecule on the surface of the local modification area 3 on the sensing substrate 2 by thiol self-assembly method or physical adsorption method; Step A2: inject the buffer solution into the reaction tank 13 of the sensor chip through the injection port 11, and measure the initial baseline output voltage through the sensor substrate 2. V b ; Step A3, injecting a solution with a target molecule concentration of [A] into the reaction tank 13 through the injection port 11 for detection, so that the target molecule binds to the probe molecule, and obtaining a binding curve of the output voltage changing with time during the binding process V m ( t ), where the relative change in output voltage during the combination process is Δ V ( t )= V m ( t )- V b ; Step A4: After reaching equilibrium during the combination process, the maximum value of the output voltage signal response is Δ V max , inject the buffer solution into the reaction tank 13 through the injection port 11, start the dissociation of the target molecule and the probe molecule, and obtain the dissociation curve of the output voltage changing with time during the dissociation process. The minimum value of the output signal response during the dissociation process is Δ Vmin ; Step A5, kinetically fitting the binding curve of step A3 and the dissociation curve of step A4 to obtain the binding rate constant k on and the dissociation rate constant k off , and calculate the equilibrium dissociation constant k D = k off / k on ; Step A6: Verification by multi-concentration target molecule experiment k obs Linear relationship with the target molecule concentration [A].

[0047] In the embodiment of the present application, in step A5, the calculation formula for kinetic fitting is as follows: ; ; .

[0048] In the embodiment of the present application, the biomolecular binding kinetics detection method is applied to the detection of antigen-antibody binding kinetics parameters, which is evolved into the following steps: Step B1, an anti-IgG antibody (concentration of 10 μg / mL) diluted with PBS buffer (pH 7.4) was immobilized on the surface of the local modified area of ​​the sensing substrate 2 by a thiol self-assembly method, and non-specific binding sites were blocked with 1% BSA; Step B2, inject PBS buffer into the reaction tank 13 of the sensor chip through the injection port 11, and control the flow rate to 5 μL / min. After the flow rate stabilizes, record the output voltage of the sensor substrate 2. V b , and the average value was taken for 5 minutes as the baseline signal; Step B3, a PBS solution containing the target antigen IgG is introduced with a concentration gradient of 0.1 nM, 1 nM, and 10 nM, and injected into the reaction tank 13 through the injection port 11 for detection, so that the anti-IgG antibody binds to the target antigen IgG, and a binding curve of the output voltage of the binding process changing with time is obtained. V m ( t ), calculate the relative change of output voltage during the combination process as Δ V ( t )= V m ( t )- Vb ; Step B4: After reaching equilibrium during the binding process, record the maximum value of the output voltage signal response as Δ V max , PBS buffer is injected into the reaction tank 13 through the injection port 11 to start the dissociation of the anti-IgG antibody and the target antigen IgG, and the dissociation curve of the output voltage changing with time during the dissociation process is recorded. The minimum output signal response value of the dissociation process is Δ V min ; Step B5, kinetically fitting the binding curve of step 3 and the dissociation curve of step 4 to obtain the binding rate constant k on and the dissociation rate constant k off , and calculate the equilibrium dissociation constant k D = k off / k on ; Step B6, repeat the above steps and measure the corresponding k obs Value, draw k obs The linear relationship between the value and the IgG concentration was plotted to calculate the error range.

[0049] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application shall be based on the protection scope of the claims.

Claims

1. A sensor chip for detecting binding kinetics between biomolecules, characterized in that: include: A control substrate, wherein at least one sample inlet and at least one sample outlet are provided on the control substrate, a reaction groove and two microchannels connected to the reaction groove are dug at the bottom end of the control substrate, and the reaction groove is connected to the sample inlet and the sample outlet respectively through the two microchannels; A sensing substrate, the top of the sensing substrate is bonded to the bottom of the control substrate, at least two first liquid accumulation grooves covered by the reaction groove are dug at the top of the sensing substrate, a structural film and a sensitive film are respectively mounted above the two first liquid accumulation grooves, a local modification area is provided at the top of the sensitive film, four cantilever beams are evenly distributed circumferentially on the side walls of the structural film and the sensitive film and are fixedly connected to the inner wall of the first liquid accumulation groove through the four cantilever beams, a piezoresistor is provided at the top of each cantilever beam, and each adjacent two piezoresistors above the same first liquid accumulation groove are connected through leads to form a Wheatstone bridge, and each lead is connected to a pad.

2. The sensor chip for detecting binding kinetics between biomolecules according to claim 1, characterized in that: Each of the leads includes two sub-leads connected to each other at one end, each of the sub-leads includes a heavily doped lead and a metal lead connected to the heavily doped lead, and each two adjacent piezoresistors above the same first liquid accumulation groove are connected in sequence through the heavily doped lead and the metal lead.

3. The sensor chip for detecting binding kinetics between biomolecules according to claim 1, characterized in that: A second liquid accumulation groove is dug at the top of each of the structural film and the sensitive film, a through hole is provided in the middle of the local modification area, and the through hole is communicated with the second liquid accumulation groove on the sensitive film.

4. The sensor chip for detecting binding kinetics between biomolecules according to claim 1, characterized in that: Four first liquid collection grooves covered by the reaction groove are dug on the top of the sensing substrate, and a structural film is mounted on the top of one of the first liquid collection grooves, and a sensitive film is mounted on the top of the other three first liquid collection grooves.

5. A method for preparing a sensor chip, characterized in that: The steps include: S1, selecting an SOI substrate including a silicon support layer, a buried oxide layer, and a silicon device layer from bottom to top; S2, sequentially performing photolithography, development and dry etching on the SOI substrate to obtain grooves on the surfaces of the silicon device layer and the buried oxide layer; S3, growing a first SiO2 layer on the surface of the groove and the silicon device layer; S4, after sequentially performing photolithography and development, lightly doping with boron ions to obtain a piezoresistor, and then heavily doping with boron ions to obtain a heavily doped lead; S5, depositing a second SiO2 layer on the top surface and the bottom surface respectively; S6, after sequentially performing photolithography, development and dry etching, a lead hole is obtained at the end of the heavily doped lead; After sequentially performing metal sputtering, photolithography, development and etching, metal leads and pads are obtained, and ohmic contact is formed between the metal leads and the heavily doped leads; S7, selectively sputtering at least one metal layer on the top surface, and obtaining a sensitive film by a metal lift-off method; S8, depositing a third SiO2 layer and a silicon nitride layer on the top surface in sequence, and then performing photolithography and development to obtain a metal passivation layer; S9, after sequentially performing photolithography, development and dry etching on the top surface, the silicon support layer is exposed and a through hole is obtained in the local modified area to obtain a second liquid storage groove; S10, performing anisotropic wet etching to obtain a first liquid accumulation groove and a cantilever beam, thereby obtaining a sensing substrate; S11, using a flexible film to control the substrate; S12, bonding the control substrate and the sensing substrate to obtain a sensor chip.

6. The preparation method according to claim 5, characterized in that: In the step S1, in the SOI substrate, the thickness of the silicon device layer is 2.9-3.1 μm, the thickness of the buried oxide layer is 0.9-1.1 μm, and the thickness of the silicon support layer is 690-710 μm.

7. The preparation method according to claim 5, characterized in that: In step S7, the material of the metal layer is a mixture of gold and chromium or a mixture of gold and titanium.

8. The preparation method according to claim 5, characterized in that: In the step S8, the thickness of the third SiO2 layer is 195-205 nm, and the thickness of the silicon nitride layer is 95-105 nm.

9. The preparation method according to claim 5, characterized in that: The specific operation of step S11 is as follows: S11, taking a single-side polished single-crystal silicon wafer, and sequentially performing photolithography and development processes to obtain a silicon wafer mold; A mixed liquid containing a flexible membrane material and a curing agent is prepared, and a silicon wafer mold is placed in the mixed liquid until it solidifies. The mold is demolded, sliced ​​and punched in sequence to obtain a control substrate with an inlet, an outlet, a reaction groove and a microfluidic channel.

10. Use of the sensor chip according to any one of claims 1 to 4 in detecting binding kinetics between biomolecules.

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